The goal of this proposal is to understand the contribution of ORAI calcium channels to cell and tissue function in health and disease. Each of the 3 ORAI proteins (ORAI1, 2, 3) forms a calcium channel when overexpressed in cells. ORAI1 is the best characterized of these and encodes the calcium release-activated calcium (CRAC). Patients with null mutations in ORAI1 suffer from severe immunodeficiency due to impaired calcium influx in immune cells. Using ORAI1 deficient mice, we found that ORAI1 is essential for lymphocyte function and immune responses in vivo and inhibition of ORAI1 protects mice from T cell mediated autoimmunity. In contrast to ORAI1, the roles of ORAI2 and ORAI3 in vivo are largely unknown and it is unclear if endogenously expressed ORAI2 and ORAI3 contribute to the function of immune or non-immune cells. This knowledge gap is in large part due to a lack of specific reagents including Orai2 and Orai3 knockout mice and high affinity antibodies to analyze the function and expression of ORAI proteins in health and disease. ORAI3 was shown to mediate calcium influx in certain cancer cells and to promote their growth and invasiveness. These findings are intriguing, but need to be corroborated using knockout mice for in vivo studies. Almost nothing is known about the role of ORAI2 in vivo. Using new Orai2-deficient reporter mice, we found expression of ORAI2 in many immune cells and the brain. In immune cells, lack of ORAI2 alone does not impair calcium influx, but its deletion together with ORAI1 abolishes calcium influx and severely impairs T cell- mediated immunity. This cooperative role of ORAI1 and ORAI2 raises the possibility that both homologs can form heteromeric channels in immune cells and potentially other cell types. To understand the physiological and pathophysiological roles of endogenous ORAI1, 2 and 3 channels in vivo we need conditional knockout mice that allow tissue and cell type-specific deletion of single or multiple Orai genes. We propose to generate conditional Orai2 and Orai3 knockout mice and to cross these to each other and Orai1-deficient mice to generate double and triple knockout mice to investigate the function of individual ORAI proteins and their synergistic roles in tissues in vivo. Conditional deletion is essential as complete deletion of Orai1 is lethal and its combined deletion with other Orai genes would therefore be lethal, too. Research on ORAI channels is severely handicapped by a lack of specific antibodies for the detection of ORAI proteins in live cells to test their expression, subcellular localization and function. We recently generated antibodies against ORAI1 that not only detect ORAI1 in live cells but also inhibit calcium influx. We propose to develop specific antibodies against ORAI2 and ORAI3 to study their expression in healthy and diseased tissues and to inhibit their function. These new tools will be shared with other labs and will provide important new insight into the physiological and pathological roles of ORAI channels, with the goal of creating rationales for the development of therapeutic ORAI channel inhibitors.